2.7 Addition of Index
Tags by
Post-hybridization
Amplification
1. SureSelect
XT Reagent Kit, MSQ, 16 (Agilent) (see Note 3).
2. PCR Indexing Primers (Agilent).
3. Postcapture PCR mix (35μL per sample): 22.5μL of NucleaseFree Water, 1μL of SureSelect GA Indexing Post-capture PCR
(forward) primer (orange cap), 10μL of Herculase II 5Â reaction buffer, 0.5μL of dNTP (100 mM), and 1μL of Herculase
II polymerase.
4. Agencourt AMPure XP beads (Beckman Coulter).
5. DynaMag-96 Side magnet (Life Technologies).
6. 70% ethanol (see Note 2).
7. Nuclease-free water.
8. Thin-wall 8-strip tubes and caps (Thermo Scientific).
9. 2200 TapeStation Nucleic Acid System (Agilent).
10. D1000 ScreenTape and D1000 reagents (Agilent).
11. Thermal cycler, e.g., SureCycler 8800, with 96-well module.
12. 5–50μL 8-channel pipette and 20–200μL 8-channel pipette.
2.8 Pooling Samples
for Multiplexed
Sequencing
1. Nuclease-free water.
2. DNA LoBind 1.5 mL tubes (Eppendorf).
3. Vortex mixer.
2.9 Illumina
Sequencing
1. MiSeq desktop sequencer (Illumina).
2. MiSeq Reagent Kit v2 (Illumina).
3. 10 M NaOH (see Note 5).
4. PhiX Control (Illumina).
5. 10 mM Tris–Cl, pH 8.5 with 0.1% Tween 20.
6. Nuclease-free water.
7. Vortex mixer.
3 Methods
3.1 Sputum
Decontamination
and DNA Extraction &
Quantification
1. Prior to treatment, all sputum specimens should be kept frozen
at À20
C.
2. Add 5 mL of N-acetyl-L-cysteine/NAOH to 5 mL of sputum.
3. Incubate tubes for 15 min at room temperature.
4. Immediately add 15 mL of PBS and centrifuge at 1865 Â g for
30 min at 4
C.
5. Discard the upper most layer and resuspend the cream layer and
pellet in 0.3 mL of sterile PBS (see Note 6).
6. Transfer the resuspended pellet (300μL, see Note 6) to a sterile
2 mL screw cap tube containing ~250μL of 0.1 mm sterile glass
beads (Becton Dickinson).
Direct WGS of M. tb’
465
Tags by
Post-hybridization
Amplification
1. SureSelect
XT Reagent Kit, MSQ, 16 (Agilent) (see Note 3).
2. PCR Indexing Primers (Agilent).
3. Postcapture PCR mix (35μL per sample): 22.5μL of NucleaseFree Water, 1μL of SureSelect GA Indexing Post-capture PCR
(forward) primer (orange cap), 10μL of Herculase II 5Â reaction buffer, 0.5μL of dNTP (100 mM), and 1μL of Herculase
II polymerase.
4. Agencourt AMPure XP beads (Beckman Coulter).
5. DynaMag-96 Side magnet (Life Technologies).
6. 70% ethanol (see Note 2).
7. Nuclease-free water.
8. Thin-wall 8-strip tubes and caps (Thermo Scientific).
9. 2200 TapeStation Nucleic Acid System (Agilent).
10. D1000 ScreenTape and D1000 reagents (Agilent).
11. Thermal cycler, e.g., SureCycler 8800, with 96-well module.
12. 5–50μL 8-channel pipette and 20–200μL 8-channel pipette.
2.8 Pooling Samples
for Multiplexed
Sequencing
1. Nuclease-free water.
2. DNA LoBind 1.5 mL tubes (Eppendorf).
3. Vortex mixer.
2.9 Illumina
Sequencing
1. MiSeq desktop sequencer (Illumina).
2. MiSeq Reagent Kit v2 (Illumina).
3. 10 M NaOH (see Note 5).
4. PhiX Control (Illumina).
5. 10 mM Tris–Cl, pH 8.5 with 0.1% Tween 20.
6. Nuclease-free water.
7. Vortex mixer.
3 Methods
3.1 Sputum
Decontamination
and DNA Extraction &
Quantification
1. Prior to treatment, all sputum specimens should be kept frozen
at À20
C.
2. Add 5 mL of N-acetyl-L-cysteine/NAOH to 5 mL of sputum.
3. Incubate tubes for 15 min at room temperature.
4. Immediately add 15 mL of PBS and centrifuge at 1865 Â g for
30 min at 4
C.
5. Discard the upper most layer and resuspend the cream layer and
pellet in 0.3 mL of sterile PBS (see Note 6).
6. Transfer the resuspended pellet (300μL, see Note 6) to a sterile
2 mL screw cap tube containing ~250μL of 0.1 mm sterile glass
beads (Becton Dickinson).
Direct WGS of M. tb’
465